Diagnosis of acute disease is often based on abnormal levels of disease markers such as enzymes and hormones in biological fluids such as serum, particularly when they change momentarily during the acute phase of disease.
The biological activity and physical properties of proteins such as enzymes and hormones is determined by structural features of the molecule, and these features are often modified by endogenous conversions factors present in body fluids. Such conversion may cause the loss of biological activity or change in physical properties such as electrophoretic mobility of the molecule. The conversion products may coexist with the original molecule immediately following the onset of an acute disease, but with the passage of time, one may find only the altered protein in the body fluids.
Many tests have been developed which measure the native form of a protein marker in a body fluid. Bioassay techniques have been traditionally used to measure enzyme activity, and when the altered form of the enzyme marker is inactive, the enzyme activity is an adequate measurement of the enzyme level in the system. Immunoassays, while offering a more convenient approach, is dependent upon having antibodies which bind selectively with the moiety to be measured. When the altered protein product differs only slightly from a native marker protein, antibodies may be unable to distinguish and will react with both moieties, giving an erroneous result. Thus the primary immunoassay efforts have addressed development of antibodies which bind specifically with the native protein marker, antibodies binding with the altered forms generally being avoided.
This invention relates to the measurement of an analyte pair consisting of the native protein and an initial, altered form and of the native protein, both measured values being useful for diagnosis. The analyte pair level remains elevated longer than the native protein, and the ratio of the amount of native analyte to the analyte pair provides a basis for more accurately estimating the time elapsed since the onset of the acute episode. In one application of this invention for the diagnosis of myocardial infarction, CK-MM isoforms are measured.
The enzyme creatine kinease (CK, ATP:creatine N-phosphotransferase) catalyzes the reversible transfer of phosphate group from ATP to creatine. It exists in the cytoplasm of the cells of human tissue as a dimer composed of two subunits commonly identified as the M-subunit and the B-subunit. Three of the commonly found isoenzymes of CK comprises combinations of these subunits and are correspondingly identified as CK-MM, CK-MB and CK-BB. Abnormality of these isoenzymes in body fluids generally suggest acute disease. CK-MB is associated with acute myocardial infarction, for example. Appearance of CK-MB isoenzymes in serum is indicative of myocardial infarction. CK-BB isoenzyme appearance in serum is indicative of brain injury and other abnormalities. CK-MM isoenzymes are present in the serum of normal patients.
Proteins such as CK-MM and CK-MB are labile and have short lives in living systems. Other endogenous enzymes act on isoenzymes and isoforms and alter their molecular size and structure. Such alteration may affect the catalytic activity, immunoreactivity and other properties. They may loose their catalytic activity and still retain immunoreactivity. They may migrate differently in separation procedures and appear in unexpected fractions. The apparent results of immunological techniques may not agree with those obtained with non-immunological techniques because of the lack of binding specificity of the antibodies. CK-BB is also believed to produce yield altered forms, but due to its short half-life and lability, no altered forms have yet been observed.
Antibodies produced with CK isoforms are frequently cross-reactive. Antibodies have been produced with CK-BB which react with CK-MB (CK-B antibodies) as reported by Shell, W. et al, "Radioimmunoassay of creatine kinease." CLINICAL NUCLEAR CARDIOLOGY. New York: Grune & Stratton pp 462-478 (1981), and antibodies have been produced with CK-MM which react with CK-MB (CK-M antibodies) as reported by Wicks, R. et al, Clin.Chem. 28:54-58 (1982). That the antibodies appear to bind with the altered proteins is also observed. Shell, W. et al, SYLLABUS. Clinical Radioassay Society, 9th Annual Meeting, p 61 (1981) speculates that an immunoassay for the CK-Bi inactive product of CK-B protein might show that the inactive form of CK-BB was present in a major proportion in serum, no CK-Bi selectively binding antibody has been reported.